Association of edg5 polymorphism v286a with type ii diabetes mellitus and venous thrombosis/pulmonary embolism and the use thereof

ABSTRACT

The present invention relates to a method of identifying an increase in risk for type II Diabetes mellitus, venous thrombosis, or pulmonary embolism in a subject, wherein the presence of an amino acid exchange at position 286 from valine (Val) to alanine (Ala) in the EDG5 protein in a biological sample taken from the subject.

FIELD OF THE INVENTION

The present invention relates generally to a method of identifying an increase in risk for type II Diabetes mellitus venous thrombosis, pulmonary embolism, or a combination thereof.

BACKGROUND OF THE INVENTION

Endothelial differentiation gene (EDG) receptors are a new family of eight G protein-coupled receptors for the lysophospholipids lysophosphatitic acid and sphingosine-1-phosphate. The lysosphingolipid sphingosine 1-phosphate (S1P) regulates cell proliferation, apoptosis, motility, and neurite retraction (Pyne and Pyne, (2000) Biochem J 349: 385-402; MacLennan et al., (2001), J. of Neurosci. 14: 203-209). Its actions may be both intracellular as a second messenger and extracellular as a receptor ligand. S1P and the structurally related lysolipid mediator lysophosphatidic acid (LPA) signal through a set of G protein-coupled receptors known as EDG receptors. EDG5 (endothelial differentiation gene 5; also termed AGR16/H218) is a functional receptor for S1P. The size of the EDG5 protein is 353 amino acids and the EDG5 gene is located on chromosome 19p13.2.

Mammalian EDG-5 Receptor homologs are described in WO 99/33972.

Developmental studies in Zebrafish have indicated that S1P signaling via the EDG5 like receptor Miles Apart is essential for heart development. The presumed function of the EDG5 homologue in Zebrafish development and its expression in the heart suggests that it may play a critical role in the development and/or function of the cardiovascular system (Kupperman et al., (2000), Nature 406: 192-195).

In order to analyze potential effects of EDG5 polymorphisms in humans, the V286A polymorphism (amino acid exchange valine to alanine at position 286 of the EDG5 protein) of the EDG5 protein was studied in a clinical patient cohort enriched for cardiovascular outcomes. So far, no data was available about the clinical effects of EDG5 variants in humans. (NCBI accession number for EDG5 protein sequence: NP_(—)004221 and NCBI accession number for EDG5 nucleotide sequence: AF034780).

The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

SUMMARY OF THE INVENTION

Broadly, the present invention extends to a method for identifying an increase in risk for type II diabetes mellitus, venous thrombosis, pulmonary embolism, or a combination thereof in a subject, comprising the steps of:

-   -   (a) removing a biological sample from the subject that comprises         an EDG5 protein; and     -   (b) determining whether the amino acid residue at position 286         of the EDG5 protein from the biological sample is Alanine.

The present invention further relates to a method for identifying an increase in risk for type II Diabetes mellitus, venous thrombosis, or pulmonary embolism in a subject, comprising the steps of:

-   -   (a) removing a biological sample from the subject that comprises         a nucleic acid sequence that encodes EDG5 protein; and     -   (b) determining whether the nucleic acid sequence encodes an         EDG5 protein comprising an alanine residue at position 286.

In a method of the present invention, the biological sample is tissue of any type, e.g. soft tissue, bone, cartilage, etc. A biological sample can also be a bodily fluid such as blood, semen, mucus, fecal material, lymph, saliva, etc.

In addition, the present invention extends to a method for selecting patients who will respond a pharmaceutical for treating type II Diabetes mellitus, venous thrombosis, or pulmonary embolism. Such a method comprises the steps of:

-   -   (a) removing a biological sample from the subject that comprises         an EDG5 protein; and     -   (b) determining whether the amino acid sequence of the EDG5         protein has an alanine residue at position 286.

Moreover, the present invention extends to a test kit for determining whether the amino acid sequence of an EDG5 protein in a biological sample taken from a subject has an alanine at position 286. Such a test kit of the present invention can be amino acid based, i.e., to evaluate the amino acid sequence of the EDG5 protein from the test sample. In a particular embodiment, the kit is an antibody-based kit. Such a kit can comprise, for example, (1) a first antibody (e.g., attached to a solid support)) that binds to an EDG5 protein having an alanine at position 286, and, optionally, (2) a second, different antibody that binds to an EDG5 protein having an alanine at position 286, or to the first antibody and is conjugated to a detectable agent. If the second antibody is not present, then either the first antibody can be detectably labeled, or alternatively, another molecule that binds the first antibody can be detectably labeled. In any event, a labeled binding moiety is included to serve as the detectable reporter molecule, as known in the art.

A kit of the present invention can also be an oligonucleotide-based kit that can be used to determine whether the biological sample comprises a nucleic acid molecule that encodes an EDG5 protein having an alanine at position 286. An oligonucleotide-based kit of the present invention can comprise, for example: (1) an oligonucleotide, e.g., a detectably-labeled oligonucleotide, that hybridizes to a nucleic acid sequence that encodes an EDG5 protein having an alanine at position 286, or (2) a pair of primers useful for amplifying a nucleic acid molecule that encodes an EDG5 protein having an alanine residue at position 286.

Naturally, a kit of the present invention can comprise, e.g., a buffering agent, a preservative or a protein stabilizing agent. The kit also can comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). Furthermore, the kit may also contain a control sample or series of control samples that can be assayed and compared to the test sample. Each component of the kit is usually enclosed within an individual container, and all of the various containers are within a single package. Instructions for observing whether the tested subject is suffering from or is at risk of type II Diabetes mellitus, venous thrombosis, or pulmonary embolism may also be enclosed.

Optionally, a kit of the present invention can also comprise directions for using the kit.

The identification of the polymorphism in the nucleotide sequence of EDG5 leading to the amino acid exchange at position 286 from Val to Ala in the EDG5 protein can be used to predict increased or normal risk for type II Diabetes mellitus and/or venous thrombosis/pulmonary embolism. It can be used e. g. in

1) methods based on sequencing the nucleotide region of interest (e.g. pyrosequencing, sequencing methods using radio-labeled nucleotides, or nucleotides which are labeled with a fluorescent dye, analysis of sequence fragments with mass spectrometry) 2) methods based on the hybridization of nucleotide sequences to the region of interest (e.g. DNA microarrays); 3) methods based on analyzing amplification products of the nucleotide region of interest (e.g. TaqMan analysis).

The identification of the polymorphism in the protein sequence of EDG5 comprising the amino acid exchange at position 286 from Val to Ala in the EDG5 protein can be used to predict increased or normal risk for type II Diabetes mellitus and/or venous thrombosis/pulmonary embolism. It can be used, e.g. in

1) methods based on sequencing the protein region of interest (e.g. standard protein degradation, analysis of protein sequence fragments with mass spectrometry); 2) methods based on using anti-EDG5 antibodies against the region of interest (e.g. ELISA);

3) methods based on analyzing functional activity of EDG5 in in-vitro assays using e. g. human, animal, bacterial, or yeast cells.

The detection of genetic polymorphisms in the EDG5 gene, in particular EDG5-286-VA (EDG5 variants having Alanine at position 286 in the protein as a consequence of pholymorphisms at the corresponding position on one allele of the EDG5 gene), and the resulting protein by analyzing human DNA or EDG5 protein may be used (a) as genetic markers for preventive treatments to type II Diabetes mellitus and/or venous thrombosis/pulmonary embolism, (b) as a genetic marker for adaptation of drug dose, (c) as a genetic marker for drug screening set-up adaptation and (d) as a genetic marker for patient selection in phase/clinical studies.

The identification of the polymorphism in the nucleotide sequence of the EDG5 gene leading to the amino acid exchange at position 286 from Val to Ala in the EDG5 protein can be used to predict increased or normal risk type II Diabetes mellitus and/or venous thrombosis/pulmonary embolism. It can be used e. g. in

1) methods based on sequencing the nucleotide region of interest (e.g. pyrosequencing, sequencing methods using radio-labeled nucleotides, or nucleotides which are labeled with a fluorescent dye, analysis of sequence fragments with mass spectrometry); 2) methods based on the hybridization of nucleotide sequences to the region of interest (e.g. DNA microarrays); 3) methods based on analyzing amplification products of the nucleotide region of interest (e.g. TaqMan analysis).

The identification of the polymorphism in the protein sequence of EDG5 comprising the amino acid exchange at position 286 from Val to Ala can be used to predict increased or normal risk for type II Diabetes mellitus and/or venous thrombosis/pulmonary embolism. It can be used, e.g. in

1) methods based on sequencing the protein region of interest (e.g. standard protein degradation, analysis of protein sequence fragments with mass spectrometry); 2) methods based on using anti-EDG5 antibodies against the region of interest (e.g. ELISA); 3) methods based on analyzing functional activity of EDG5 in in-vitro assays using e. g. human, animal, bacterial, or yeast cells.

The detection of genetic polymorphisms in the EDG5 gene, in particular EDG5-286-VA (EDG5 variants having Alanine at position 286 of the protein as a consequence of pholymorphisms at the corresponding position on one allele of the EDG5 gene), and the resulting protein by analyzing human DNA and EDG5 protein may be used (a) as genetic markers for preventive treatments to prevent type II Diabetes mellitus and/or venous thrombosis/pulmonary embolism, (b) as a genetic marker for adaptation of drug dose, (c) as a genetic marker for drug screening set-up adaptation and (d) as a genetic marker for patient selection in phase/clinical studies.

The present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following Examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

The known EDG5 polymorphism at the position 286 of the EDG5 protein (NCBI accession number for protein sequence: NP_(—)004221 (Table 1A); NCBI accession number for nucleotide sequence: AF034780 (Table 1 B)) was analyzed in a patient cohort with or without cardiovascular events or endpoints.

Example 1

Study Subjects (Study Population):

The genomic DNA of 1140 patients was screened for single nucleotide polymorphisms (SNPs) in the EDG5 gene leading to the protein variants EDG5-V286A. The phenotype of this patient cohort has been described previously (Winkelmann et al. (2001) Pharmacogenomics, 2, 1-73). Inclusion criteria have been: Caucasian individual of German ancestry, stable clinical condition (except for acute coronary syndrome [ACS]) and coronary angiogram. Exclusion criteria have been: acute illness other than ACS, chronic non-cardiac disease (i.e. rheumatic arthritis) and history of malignant disease within the previous five years. Basic characteristics of this patient cohort are outlined in Table 2.

Example 2

SNP Detection by Sequencing and Analysis

Example 2.1

Amplification of Genomic Region with Polymorphism of Interest

Amplification Primers:

1. For the detection of nucleotide exchange of valine to alanine at position 286 of the EDG5 gene sequence, the following primers were used:

(SEQ ID NO. 1) Forward primer : 5′-TCCACTGTCCTGCCTCTCTAC-3′ (SQE ID NO. 2) Reverse primer : 5′-TCTCCATGAACCCCTCTGCC-3′

PCR Protocol for Amplification:

All reagents are from Applied Biosystems (Foster City, USA): 20 ng of genomic DNA; 1 unit of TaqGold polymerase; 1× Taq polymerase buffer; 500 μM of dNTP; 2,5 mM of

MgCl2; 200 nM of each amplification primer pair (for sequence see Amplification primer pair 1. above); H₂O ad 5 μl.

Amplification Program for PCR/Genotyping:

-   95° C.×10 min×1 cycle -   95° C.×30 sec -   70° C.×30 sec×2 cycles; -   95° C.×30 sec -   65° C.×30 sec×2 cycles; -   95° C.×30 sec -   60° C.×30 sec×2 cycles; -   95° C.×30 sec -   56° C.×30 sec -   72° C.×30 sec×40 cycles; -   72° C.×10 min -   4° C.×30 sec×1 cycle;

Example 2.2

Identification of Polymorphisms of Interest

Protocol for Minisequencing and Detection of Polymorphisms:

All reagents are from Applied Biosystems (Foster City, USA). 2 μl of purified PCR product; 1.5 μl BigDye terminator kit; 200 nM of one sequencing primer (for sequence see forward or reverse Amplification primer 1. above); H₂O ad 10 μl.

Amplification Program for Sequencing:

-   96° C.×2′×1 cycle; -   96° C.×10″ -   55° C.×10″ -   65° C.×4′×30 cycles; -   72° C.×7′ -   4° C.×30″×1 cycle;

Analysis of Sequencing Products:

Sequences were analyzed first with sequencing analysis (Applied Biosystems, Foster City, USA) for raw data extraction, then processed with PHRED (base caller), PHRAP (assembler), POLYPHRED (SNP caller) and CONSED (results viewer). PHRED, PHRAP, POLYPHRED and CONSED are software designed at Washington University by Phil Green (http://www.genome.washington.edu).

Example 2.3

Statistical Approaches for Genotype/Phenotype Correlation

All analyses were done with SAS statistical package (Version 6.12, SAS Institute GmbH, Heidelberg/Germany). For the detection of associations between genetic polymorphisms and a large number of clinical relevant parameters, descriptive statistics were computed (median, quartiles) and Wilcoxon-rank-sum-tests were performed. Wilcoxon-rank-sum-test is used for the comparison of two independent samples. The computation of the test statistic is based on ranks in the pooled sample.

The search for associations between the SNPs and risk factors and diseases was done in a similar way. The Chi-Square-Test was performed and numbers and percentages were calculated to describe the data. The Chi-Square-Test is a statistical test for calculating the dependence of two variables. The values of the variables are contained in two or more classes. To analyze the association of those variables, a contingency table is used. This table contains as many rows as the number of realizations of the first variable and as many columns as the number of realizations of the second variable. Every cell contains a special patient's characteristic. To construct a test statistic, the differences of calculated and observed frequencies are computed.

After inspecting the results, relevant variables were selected. To take account of confounding co-variables, logistic regression was used to validate the results. The logistic regression method is used to analyze the influence of several explanatory variables on a certain response variable. The associated statistical test gives a p-value. The interpretation of this p-value is that there is a significant influence of the associated explanatory variable.

For a binary variable, the odds ratio has been calculated. The odds ratio is the ratio of the odds that an event will occur in one group to the odds that the event will occur in the other group.

Example 2.4

Analyses

The following abbreviations are used:

EDG5-286-W defines the group of individuals, in which both of the EDG5 alleles code for a EDG5 gene variant leading to Valine (V) at position 286 of the EDG5 protein, this group is homozygous for this EDG5 polymorphism at position 286 of the EDG5 protein.

EDG5-286-VA defines the group of individuals, in which one of the EDG5 alleles codes for a EDG5 gene variant leading to Valine (V) at position 286 of the EDG5 protein and the other EDG5 allele codes for a EDG5 gene variant leading to Alanine (A) at position 286 of the EDG5 protein, this group is heterozygous for EDG5 polymorphism at position 286 of the EDG5 protein.

EDG5-286-AA defines the group of individuals, in which both of the EDG5 alleles code for a EDG5 gene variant leading to Alanine (A) at position 286 of the EDG5 protein, this group is homozygous for this EDG5 polymorphism at position 286 of the EDG5 protein.

The distribution of EDG5-286 variants in 1140 individuals is shown in Table 3. The EDG5-286-AA variant does not appear at all in this patient cohort. 2.1% of all patients are carriers of the EDG5-286-VA variant and 97.8% of the patients are carriers of the EDG5-286-W variant.

Patients carrying EDG5-286-VA show an increased association for type II diabetes mellitus (DM Type II) and venous thrombosis/pulmonary embolism (VT/PE) compared to EDG5-286-VV patients. Statistical significance calculated with Chi-square test of the observed association is p-value =0.001 for the association with DM Type II and p-value =0.026 for the association with VT/PE (Tables 4A and 5A). Logistic regression models for analyzing the influence of confounding factors, such as myocardial infarction and hypertension resulted in a p-value=0.0022 for the association of EDG5-286-VA with DM Type II to p-value and p-value=0.0315 for the association of EDG5-286-VA with VT/PE (Tables 4B and 5B).

The odds ratios of decreased risk for DM Type II is 3.801 and for VT/PE 3.095 in individuals carrying the EDG5-286-VA variants compared to individuals with EDG5-286-W variants (Tables 4C and 5C).

The EDG5-286-VA allele represents therefore a strong genetic marker to estimate decreased risk of DM Type II and venous thrombosis/pulmonary embolism.

Table 1:

Protein sequence of EDG5 (ENDOTHELIAL DIFFERENTIATION GENE 5). The protein sequence accession number (NCBI protein database) of EDG5 is NP_(—)004221 (A), the nucleotide sequence accession number (NCBI nucleotide database) is AF034780 (B) and the accession number for EDG5 information in OMIM (ONLINE MENDELIAN INHERITANCE IN MAN) is 605111.

TABLE 1A Protein Sequence of EDG5 (SEQ ID NO. 3) 1 MGSLYSEYLN PNKVQEHYNY TKETLETQET TSRQVASAFI VILCCAIVVE NLLVLIAVAR 61 NSKFHSAMYL FLGNLAASDL LAGVAFVANT LLSGSVTLRL TPVQWFAREG SASITLSASV 121 FSLLAIAIER HVAIAKVKLY GSDKSCRMLL LIGASWLISL VLGGLPILGWNCLGHLEACS 181 TVLPLYAKHY VLCVVTIFSI ILLAIVALYV RIYCVVRSSH ADMAAPQTLALLKTVTIVLG 241 VFIVCWLPAF SILLLDYACP VHSCPILYKA HYFFAVSTLN SLLNPVIYTWRSRDLRREVL 301 RPLQCWRPGV GVQGRRRVGT PGHHLLPLRS SSSLERGMHM PTSPTFLEGN TVV

TABLE 1B Nucleotide Sequence of EDG5 (SEQ ID NO. 4) 1 ATGGGCAGCT TGTACTCGGA GTACCTGAAC CCCAACAAGG TCCAGGAACA CTATAATTAT 61 ACCAAGGAGA CGCTGGAAAC GCAGGAGACG ACCTCCCGCC AGGTGGCCTC GGCCTTCATC 121 GTCATCCTCT GTTGCGCCAT TGTGGTGGAA AACCTTCTGG TGCTCATTGC GGTGGCCCGA 181 AACAGCAAGT TCCACTCGGC AATGTACCTG TTTCTGGGCA ACCTGGCCGC CTCCGATCTA 241 CTGGCAGGCG TGGCCTTCGT AGCCAATACC TTGCTCTCTG GCTCTGTCAC GCTGAGGCTG 301 ACGCCTGTGC AGTGGTTTGC CCGGGAGGGC TCTGCCTCCA TCACGCTCTC GGCCTCTGTC 361 TTCAGCCTCC TGGCCATCGC CATTGAGCGC CACGTGGCCA TTGCCAAGGT CAAGCTGTAT 421 GGCAGCGACA AGAGCTGCCG CATGCTTCTG CTCATCGGGG CCTCGTGGCT CATCTCGCTG 481 GTCCTCGGTG GCCTGCCCAT CCTTGGCTGG AACTGCCTGG GCCACCTCGA GGCCTGCTCC 541 ACTGTCCTGC CTCTCTACGC CAAGCATTAT GTGCTGTGCG TGGTGACCAT CTTCTCCATC 601 ATCCTGTTGG CCATCGTGGC CCTGTACGTG CGCATCTACT GCGTGGTCCG CTCAAGCCAC 661 GCTGACATGG CCGCCCCGCA GACGCTAGCC CTGCTCAAGA CGGTCACCAT CGTGCTAGGC 721 GTCTTTATCG TCTGCTGGCT GCCCGCCTTC AGCATCCTCC TTCTGGACTA TGCCTGTCCC 781 GTCCACTCCT GCCCGATCCT CTACAAAGCC CACTACTTTT TCGCCGTCTC CACCCTGAAT 841 TCCCTGCTCA ACCCCGTCAT CTACACGTGG CGCAGCCGGG ACCTGCGGCG GGAGGTGCTT 901 CGGCCGCTGC AGTGCTGGCG GCCGGGGGTG GGGGTGCAAG GACGGAGGCG GGTCGGGACC 961 CCGGGCCACC ACCTCCTGCC ACTCCGCAGC TCCAGCTCCC TGGAGAGGGG CATGCACATG 1021 CCCACGTCAC CCACGTTTCT GGAGGGCAAC ACGGTGGTCT GA

TABLE 2 Basic characteristics of the patient cohort. n % Total 1140 Gender Female 782 68.6 Male 358 31.4 Age* 63.3 (56.5-70.9) BMI* 27.9 (Body Mass (25.0-30.3) Index) Hypertension 670 58.8 Smoker 731 64.1 Diabetes no oGT 279 24.5 normal GT 264 23.2 impaired GT 236 20.7 diabetes new 169 14.8 known diabetes 192 16.8 Diabetes diabetes (total) 361 31.7 no diabetes (total) 779 68.3 Stable CCS 1 457 40.1 CAD CCS 2 409 35.9 CCS 3 191 16.8 CCS 4 83 7.3 Unstable no ACS (noCAD/stable CAD/MI > 743 65.2 CAD 15 d) tropT − UA (no acute MI) 250 21.9 tropT + UA (no clinical MI) 42 3.7 post acute MI (1-15 d) 105 9.2 *Median and Quartiles (Q1-Q3)

TABLE 3 Distribution of EDG5 variants in the analyzed patient cohort. Number of patients (%) EDG5-286-VV 1116 (97.9%) EDG5-286-VA 24 (2.1%) EDG5-286-AA 0 (0%)

TABLE 4A Association of EDG5-286 variants with type II diabetes mellitus calculated by Chi-square test. Type II Diabetes Mellitus Number of patients Number of patients with p- without T-II-DM (%) T-II-DM (%) value EDG5-286-VA 9 (37.5%) 15 (62.5%) 0.001 EDG5-286-VV 770 (69.0%) 346 (31.0%) An increased frequency of EDG5-286-VA carriers in the type II diabetes mellitus positive group could be observed compared to EDG5-286-VV patients.

TABLE 4B Calculation of statistical significance for EDG5-286-VA association with type II diabetes mellitus by logistic regression. p-value (logistic regression) EDG5-286-VA 0.0022 Male gender 0.9926 Smoker 0.7159 Arterial hypertension 0.0001 MI 0.0044 ACS 0.5657 Tot. Cholesterol >=240 or drug history 0.4831 Venous thrombosis/pulmonary 0.5843 embolism

TABLE 4C Calculation of odds ratios for the risk of having type II diabetes mellitus of EDG5-286-VA patients compared to EDG5-286-VV patients. 95%-confidence interval odds ratio lower Upper p-value Type II Diabetes Mellitus 3.801 1.614 8.949 0.0022

TABLE 5A Association of EDG5-286 variants with venous thrombosis/pulmonary embolism calculated by Chi-square test. Venous thrombosis/pulmonary embolism (VT/PE) Number of patients Number of patients p- without VT/PE (%) with VT/PE (%) value EDG5-286-VA 19 (79.2%) 5 (20.8%) 0.026 EDG5-286-VV 1030 (93.0%) 78 (7.0%) An increased frequency of EDG5-286-VA carriers in the venous thrombosis/pulmonary embolism positive group could be observed compared to EDG5-286-VV patients.

TABLE 5B Calculation of statistical significance for EDG5- 286-VA association venous thrombosis/pulmonary embolism (VT/PE) by logistic regression. p-value (logistic regression) EDG5-286-VA 0.0351 Male gender 0.0004 Smoker 0.0965 Arterial hypertension 0.7338 MI 0.8597 ACS 0.9270 Diabetes mellitus type II 0.5796 Tot. Cholesterol >=240 or drug history 0.9452

TABLE 5C Calculation of odds ratios for the risk of having venous thrombosis/pulmonary embolism of EDG5-286-VA patients compared to EDG5-286-VV patients. 95%-confidence interval odds ratio lower Upper p-value Venous thrombosis/ 3.095 1.082 8.853 0.0351 Pulmonary embolism

The present invention is not to be limited in scope by the specific embodiments describe herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. 

1. A test kit for testing for the presence in a biological sample taken from a subject of a DNA sequence that encodes an EDG5 protein having an alanine residue at position 286, comprising: (a) PCR oligonucleotide primers suitable for detection of the DNA sequence; and (b) other reagents.
 2. The test kit of claim 2, further comprising directions for using the kit.
 3. A commercial test kit for detecting the presence in a biological sample taken from a subject of an EDG5 protein having an alanine at position 286, comprising (a) a predetermined amount of at least one detectably labeled immunochemically reactive component having affinity for the EDG5 protein having an alanine at position 286; and (b) other reagents.
 4. The test kit of claim 3, further comprising directions for use of the kit. 